Project Summary. Major depressive disorder (MDD) is among the most common psychiatric illnesses in the United States and worldwide, contributing significantly to the morbidity and lost quality of life attributable to mental illness. In spite of personal and societal impacts of MDD, little progress has been made with regards to its cellular and brain- wide etiology or pathogenesis?except, notably, replicable findings of lost hippocampal volume in patients. This paucity of scientific information leaves additional questions as to why females experience MDD twice as often. MDD-associated genetic regions recently identified in genome-wide association studies (GWAS) are a first step toward identifying candidate genes and regulators underlying MDD and its sex differential incidence. However, regions identified in GWAS, including those of MDD, generally contain tens or hundreds of single- nucleotide variants in linkage, the vast majority being noncoding and regulatory in nature. Thus, attributing biological function to a disease-associated GWAS region requires experimental identification of functional? that is, gene-expression-altering?SNPs. Moreover, identifying functional SNPs requires study in disease relevant cell-types, as regulatory elements behave in cell-type specific manners. A recent MDD GWAS identified over a dozen loci, including ones near the genes Slc6a15 and Lin28b, which have existing biological evidence to support their role in MDD. SNPs near Slc6a15 have been attributed to sex differential volume changes in hippocampus in MDD patients, and Lin28b has been implicated by dysregulation of its targets, let-7 miRNAs, in depression patients, mouse models; Lin28b is also implicated in hippocampal neurodegeneration. Therefore, this project seeks to thoroughly characterize sequences regulating these two genes, including sex- specific regulators, and further seeks to identify functional SNPs from these two human GWAS loci. Using massively-parallel reporter assays (MPRA), short 140bp regions of genomic sequences are paired to unique 9bp barcodes and a reporter gene. Transfection of the entire library of custom sequences, followed by RNA sequencing, allows quantification of transcribed barcodes, each of which defines a specific 140bp sequence. This allows for quantitative comparison of gene expression, including between sexes for the same sequence and between SNP alleles. To study regulation of Slc6a15 and Lin28b, these experiments will be performed in mouse neurospheres (for mouse genomic sequences near the two genes) and human neural progenitor cells (for human genomic regions containing the linked SNPs). To assay these elements in a mature CNS context, we also will deliver these MPRA libraries in vivo to the mouse brain, and selectively retrieve neuronal mRNAs (i.e., including barcoded reporter RNAs) from the hippocampus using translating ribosome affinity purification (TRAP). Ultimately, these findings will begin to unravel sex-differential gene regulation in the brain and identify functional SNPs in MDD GWAS loci.